Patent Application
20220127640
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5576-387_ST25.txt, 7,988 bytes in size, generated on Jul. 22, 2021, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
The present invention relates to artificial microRNA precursors and to improved microRNA expression vectors containing the same.
RNA interference (RNAi) is a phenomenon in which a small double-stranded RNA (siRNA) consisting of about 21 base pairs causes post-transcriptional gene silencing. To supply siRNA stably in cells, a short hairpin RNA (shRNA) expression vector is usually introduced into cells. However, unphysiological overexpression of shRNA is known to cause cytotoxicity by saturating and inhibiting the endogenous microRNA (miRNA) production pathway. To avoid this problem, development of an artificial miRNA expression vector utilizing the stem-loop structure of endogenous miRNA precursors has been attempted, and as such vectors, DNA plasmid vectors and retroviruses are frequently used (Non-Patent Documents 1, 2). However, these vectors intrude into nuclei and express primary transcription products of miRNA, and hence have a risk of causing incorporation into chromosomal DNA of host cells.
On the other hand, the present inventors have developed a cytoplasmic RNA vector based on Sendai virus (SeV), which can express a foreign gene in the cytoplasm with a high efficiency without entering nuclei (Patent Documents 1, 2). This vector allows simultaneous, stable expression of multiple foreign genes from a single vector, and it has no risk of damaging chromosomal DNA of host cells, and thus, has low cytotoxicity. Accordingly, the vector is particularly suitable for production of iPS cells (Patent Document 3) and are currently used for stem cell research, etc., in many laboratories in Japan and abroad. If the vector is useful in the expression of artificial miRNA, it can serve as an excellent tool for gene silencing, and it is expected to contribute to a wide variety of studies including fundamental research and applied research. However, cytoplasmic RNA vectors cannot use the intranuclear miRNA production pathway, and hence suffer from a problem of low miRNA expression efficiency.
The present invention has been made for the purpose of providing a vector that has low cytotoxicity and is capable of expressing artificial miRNA/siRNA with a high efficiency without adverse influence on host cells.
The present inventors have earnestly researched, and as a result, have succeeded in expressing artificial miRNA/siRNA from various virus vectors or nonviral vectors by using an artificial microRNA precursor based on the structure of an miR-367 precursor.
Specifically, according to one embodiment, the present invention provides an isolated RNA molecule comprising an artificial microRNA precursor comprising in the 5′ 3′ direction: a first terminal oligonucleotide; a passenger strand oligonucleotide; a first central oligonucleotide consisting of CYG (SEQ ID NO: 2), wherein Y is C or U; a second central oligonucleotide consisting of a nucleotide sequence having at least 70% homology with UUGAAUAKAAAU (SEQ ID NO: 3), wherein K is G or U; a third central oligonucleotide consisting of YGG (SEQ ID NO: 4), wherein Y is C or U; a guide strand oligonucleotide; and a second terminal oligonucleotide; wherein the guide strand oligonucleotide consists of 17 to 29 nucleotides having complementarity to a target sequence in an mRNA of a target gene; wherein the passenger strand oligonucleotide has a length identical to the length of the guide strand oligonucleotide or has a length one to three nucleotides shorter than the length of the guide strand oligonucleotide; wherein the first terminal oligonucleotide consists of AGGCCR (SEQ ID NO: 1) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 1 are substituted, wherein R is A or G; wherein the second terminal oligonucleotide consists of UGGAYYK (SEQ ID NO: 5) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 5 are substituted, wherein Y is C or U independently on each occurrence and K is G or U; wherein the first terminal oligonucleotide and the second terminal oligonucleotide pair to form a first structural stem region; wherein the passenger strand oligonucleotide and the guide strand oligonucleotide pair to form a double-stranded microRNA region; wherein the first central oligonucleotide and the third central oligonucleotide pair to form a second structural stem region; wherein the first structural stem region, the double-stranded microRNA region, and the second structural stem region together form a stem structure; and wherein the second central oligonucleotide forms a loop structure.
The double-stranded microRNA region may include a mismatch or a bulge.
A spacer oligonucleotide consisting of 1 to 10 nucleotides may be further comprised between the first central oligonucleotide and the second central oligonucleotide, or between the second central oligonucleotide and the third central oligonucleotide.
According to one embodiment, the present invention provides an expression vector comprising: the above isolated RNA molecule or an RNA molecule consisting of a complementary sequence thereto, or a DNA molecule coding therefor.
The expression vector is preferably an RNA virus vector, is more preferably a cytoplasmic RNA virus vector, and is particularly preferably a Sendai virus vector.
The isolated RNA molecule comprising an artificial microRNA precursor according to the present invention can express artificial miRNA/siRNA with a high efficiency not only from conventional DNA plasmid vectors but also from cytoplasmic RNA virus vectors. Therefore, when used in combination with a cytoplasmic RNA virus vector, the isolated RNA molecule can express artificial miRNA/siRNA with a high efficiency without adverse influence on host cells, thus being useful.
Hereinafter, the present invention will be described in detail; however, the present invention is not limited to embodiments described herein.
According to the first embodiment, the present invention is an isolated RNA molecule comprising an artificial microRNA precursor comprising in the 5′ 3′ direction: a first terminal oligonucleotide; a passenger strand oligonucleotide; a first central oligonucleotide consisting of CYG (SEQ ID NO: 2), wherein Y is C or U; a second central oligonucleotide consisting of a nucleotide sequence having at least 70% homology with UUGAAUAKAAAU (SEQ ID NO: 3), wherein K is G or U; a third central oligonucleotide consisting of YGG (SEQ ID NO: 4), wherein Y is C or U; a guide strand oligonucleotide; and a second terminal oligonucleotide; wherein the guide strand oligonucleotide consists of 17 to 29 nucleotides having complementarity to a target sequence in an mRNA of a target gene; wherein the passenger strand oligonucleotide has a length identical to the length of the guide strand oligonucleotide or has a length one to three nucleotides shorter than the length of the guide strand oligonucleotide; wherein the first terminal oligonucleotide consists of AGGCCR (SEQ ID NO: 1) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 1 are substituted, wherein R is A or G; wherein the second terminal oligonucleotide consists of UGGAYYK (SEQ ID NO: 5) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 5 are substituted, wherein Y is C or U independently on each occurrence and K is G or U; wherein the first terminal oligonucleotide and the second terminal oligonucleotide pair to form a first structural stem region; wherein the passenger strand oligonucleotide and the guide strand oligonucleotide pair to form a double-stranded microRNA region; wherein the first central oligonucleotide and the third central oligonucleotide pair to form a second structural stem region; wherein the first structural stem region, the double-stranded microRNA region, and the second structural stem region together form a stem structure; and wherein the second central oligonucleotide forms a loop structure.
In the present embodiment, “isolated” means that the RNA molecule according to the present embodiment is in a state purified so as to contain substantially no other nucleic acids; specifically, so that the RNA molecule according to the present embodiment has a purity of at least 90%, preferably at least 95%, and particularly preferably 99% or higher.
In the present embodiment, “artificial microRNA precursor” refers to an unnatural RNA molecule that mimics the structure of a known or wild-type microRNA (hereinafter, also referred to as “miRNA”) precursor and expresses natural or artificial miRNA or siRNA. The scope of the artificial miRNA precursor according to the present embodiment can include both pri-miRNA and pre-miRNA.
The artificial miRNA precursor according to the present embodiment includes, as a first component, a first structural stem region formed by pairing of a first terminal oligonucleotide and a second terminal oligonucleotide. Here, “pair(ing)” means formation of base pairs between two oligonucleotides, and these base pairs may include not only G:C and A:U but also wobble base pairs (G:U). In the present embodiments, the first structural stem region in the artificial miRNA precursor is based on the structures of mouse and human miR-367 precursors, and may be completely identical or substantially the same as the corresponding part of the structure of a mouse or human miR-367 precursor. “Substantially the same” means that nucleotide substitutions are included to a degree that does not affect the entire structure of the stem region (e.g., about one to three nucleotide substitutions).
Specifically, the first terminal oligonucleotide consists of AGGCCR (SEQ ID NO: 1) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 1 are substituted, and the second terminal oligonucleotide consists of UGGAYYK (SEQ ID NO: 5) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 5 are substituted. Here, R is A or G in the nucleotides of SEQ ID NO: 1, and Y is C or U independently on each occurrence and K is G or U in the nucleotides of SEQ ID NO: 5. Positions and types of nucleotide substitutions are not particularly limited as long as the entire structure of the first structural stem region is retained. Preferably, the first terminal oligonucleotide may consist of AGGCCG (SEQ ID NO: 6) or AGGCCA (SEQ ID NO: 7), and the second terminal oligonucleotide may consist of UGGACCU (SEQ ID NO: 8) or UGGAUUG (SEQ ID NO: 9).
The artificial miRNA precursor according to the present embodiment includes, as a second component, a double-stranded microRNA region formed by pairing of a passenger strand oligonucleotide and the guide strand oligonucleotide. In double-stranded miRNA, “guide strand” refers to a strand that becomes mature miRNA (i.e., an antisense strand in siRNA), and “passenger strand” refers to a strand that is to be removed from double-stranded miRNA and decomposed (i.e., a sense strand in siRNA).
In the present embodiment, the guide strand oligonucleotide consists of 17 to 29 nucleotides, preferably 19 to 25 nucleotides, particularly preferably 21 to 23 nucleotides, and most preferably 22 nucleotides, having complementarity to a target sequence in an mRNA of a target gene. A target sequence in an mRNA of a target gene can be appropriately selected so that expression of the target gene can be specifically suppressed with an already established designing method for artificial miRNA/siRNA in the art.
To completely suppress expression of a target gene, it is preferable that the guide strand oligonucleotide according to the present embodiment consist of a sequence having perfect or complete (i.e., 100%) complementarity to the target sequence; to suppress expression of a target gene to a low to medium degree, it may be sufficient to use a sequence having complementarity to a degree that allows specific recognition of an mRNA of the target gene. Hence, it may be sufficient for the guide strand oligonucleotide according to the present embodiment to have at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence complementarity to a target sequence in an mRNA of a target gene. In other words, the guide strand oligonucleotide according to the present embodiment may have, for example, a sequence such that 10 or fewer, eight or fewer, six or fewer, five or fewer, four or fewer, three or fewer, two, or one nucleotide substituted in a sequence completely complementary to a target sequence in an mRNA of a target gene. Sequence complementarity can be calculated by using any calculation algorithm conventionally used in the art (e.g., NCBI BLAST).
In the present embodiment, the passenger strand oligonucleotide has a length identical to the length of the guide strand oligonucleotide or has a length one to three nucleotides shorter than the length of the guide strand oligonucleotide. Thus, it follows that if the guide strand oligonucleotide consists of 22 nucleotides, the passenger strand oligonucleotide consists of 19 to 22 nucleotides. In the present embodiment, it is preferable that the passenger strand oligonucleotide have 100% complementarity to the guide strand oligonucleotide; however, the passenger strand oligonucleotide may include, for example, one, two, three, four, or five mismatches or bulges, as long as the passenger strand oligonucleotide and the guide strand oligonucleotide can pair to form a double strand. The position of a mismatch or bulge may be arbitrary, and can be preferably a position corresponding to a mismatch or bulge in mouse and human miR-367 precursors, and specifically the 2-position, the 8-position, and/or the 9-position from the 5′ end of the guide strand oligonucleotide are/is preferred.
The artificial miRNA precursor according to the present embodiment includes, as a third component, a second structural stem region formed by pairing of a first central oligonucleotide consisting of CYG (SEQ ID NO: 2) and a third central oligonucleotide consisting of YGG (SEQ ID NO: 4). Here, Y is C or U independently in each occurrence in the nucleotides of SEQ ID NOs: 2 and 4. In the present embodiment, the second structural stem region in the artificial miRNA precursor is based on the structures of mouse and human miR-367 precursors, and it may be completely identical or substantially the same as the corresponding part of the structure of a mouse or human miR-367 precursor. Preferably, the first central oligonucleotide may consist of CUG (SEQ ID NO: 10), and the third central oligonucleotide may consist of UGG (SEQ ID NO: 11).
In the artificial miRNA precursor according to the present embodiment, the first structural stem region, the double-stranded miRNA region, and the second structural stem region together form a stem structure. Here, the stem structure may consist only of the first structural stem region, the double-stranded miRNA region, and the second structural stem region, or may include nucleotide insertions between the first structural stem region and the double-stranded miRNA region and/or between the double-stranded miRNA region and the second structural stem region to a degree that does not affect the entire stem structure. Specifically, a few (e.g., one, two, or three) nucleotide insertions may exist between the first terminal oligonucleotide and the passenger strand oligonucleotide, between the passenger strand oligonucleotide and the first central oligonucleotide, between the third central oligonucleotide and the guide strand oligonucleotide, and/or between the guide strand oligonucleotide and the second terminal oligonucleotide. In the present embodiment, it is preferable that the stem structure of the artificial miRNA precursor be composed only of the first structural stem region, the double-stranded miRNA region, and the second structural stem region directly linked together.
The artificial miRNA precursor according to the present embodiment includes, as a fourth component, a second central oligonucleotide consisting of a nucleotide sequence having at least 70% homology with UUGAAUAKAAAU (SEQ ID NO: 3). Here, K is G or U in the nucleotides of SEQ ID NO: 3. The second central oligonucleotide according to the present embodiment is based on the structures of mouse and human miR-367 precursors, and may be in any mode that allows formation of a loop structure similar to those of mouse and human miR-367 precursors. Thus, the second central oligonucleotide according to the present embodiment may consist of a nucleotide sequence having at least 70% or 80% homology with a nucleotide sequence consisting of UUGAAUAKAAAU (SEQ ID NO: 3), and may consist of a nucleotide sequence preferably having at least 90% or higher homology, particularly preferably having 100% homology, with SEQ ID NO: 3. In other words, the second central oligonucleotide according to the present embodiment may have a nucleotide sequence such that, for example, four or fewer, three or fewer, two, or one nucleotide is substituted, deleted, or inserted in a nucleotide sequence consisting of UUGAAUAKAAAU (SEQ ID NO: 3). Preferably, the second central oligonucleotide may consist of UUGAAUAGAAAU (SEQ ID NO: 12) or UUGAAUAUAAAU (SEQ ID NO: 13).
In the present embodiment, it is preferable that the first central oligonucleotide, the second central oligonucleotide, and the third central oligonucleotide be directly linked together; however, a spacer oligonucleotide consisting of 1 to 10 nucleotides, one to five nucleotides, or one to three nucleotides may be included between the first central oligonucleotide and the second central oligonucleotide, or between the second central oligonucleotide and the third central oligonucleotide. The spacer oligonucleotide may have any sequence, but it is preferable for the spacer oligonucleotide to have a sequence that does not form base pairs with the nucleotide sequence consisting of UUGAAUAKAAAU (SEQ ID NO: 3).
The isolated RNA molecule according to the present embodiment may be prepared with optional addition of a flanking sequence to the 5′ end and/or 3′ end of an artificial miRNA precursor designed according to the above description. Such a flanking sequence can be appropriately determined according to an expression vector into which the isolated RNA molecule is incorporated. The flanking sequence may include a sequence corresponding to the flanking sequence of a natural miR-367 precursor, and the length may be, for example, 1 to 100 nucleotides, 1 to 50 nucleotides, or 1 to 40 nucleotides, and may be preferably 15 to 25 nucleotides.
The isolated RNA molecule according to the present embodiment can be chemically synthesized or biosynthesized through a gene engineering procedure by using a method known in the art. For example, the isolated RNA molecule according to the present embodiment can be produced through preparation of template DNA followed by transcription thereof with RNA polymerase. The isolated RNA molecule according to the present embodiment may be composed totally of RNA or contain in part modified RNA. Examples of modified RNA include phosphorothioated RNA, boranophosphated RNA, 2′-O-methylated RNA, 2′-F RNA, and 2′,4′-BNA (also known as: LNA (Locked Nucleic Acid)).
Artificial miRNA/siRNA can be expressed by introducing the isolated RNA molecule according to the present embodiment or a DNA molecule encoding thereof into cells. Introduction of the RNA molecule or the DNA molecule into cells can be performed by using a well-known method in the art according to the type of cell, for example, by lipofection, microinjection, or electroporation.
Alternatively, artificial miRNA/siRNA can be expressed with a high efficiency by incorporating the isolated RNA molecule according to the present embodiment into various expression vectors, including cytoplasmic RNA virus vectors, and then introducing the result into a cell.
Hence, according to the second embodiment, the present invention is an expression vector comprising the above isolated RNA molecule, an RNA molecule consisting of a complementary sequence thereto, or a DNA molecule encoding the isolated RNA molecule or the RNA molecule.
The expression vector applicable in the present embodiment is not limited to particular types, and both virus vectors and nonviral vectors can be used. Examples of virus vectors include, but are not limited to, DNA virus vectors such as adenovirus vectors, adeno-associated virus vectors, and herpesvirus vectors, and RNA virus vectors such as retrovirus vectors, lentivirus vectors, bornavirus vectors, and paramyxovirus vectors. Examples of nonviral vectors include plasmid vectors such as pOL1 (produced in Examples below), pCI Mammalian Expression Vector (Promega Corporation), and pBApo-CMV DNA (Takara Bio Inc.), and episomal vectors such as pEBMulti-Hyg (FUJIFILM Corporation).
The expression vector according to the present embodiment is preferably an RNA virus vector, and more preferably a cytoplasmic RNA virus vector. The cytoplasmic RNA virus vector may be selected from, for example, paramyxovirus vectors such as Sendai virus vectors, alphavirus vectors such as Sindbis virus vectors, flavivirus vectors such as tick-borne encephalitis virus vectors, and vesiculovirus vectors such as vesicular stomatitis virus vectors. The expression vector according to the present embodiment can be particularly preferably a Sendai virus vector.
The expression vector according to the present embodiment can be prepared by operably linking the above isolated RNA molecule or an RNA molecule consisting of a complementary sequence thereto, or a DNA molecule encoding the isolated RNA molecule or the RNA molecule to the downstream of a promoter in an expression vector. One or more isolated RNA molecules, or DNA molecules coding therefor as defined above, may be introduced into a single expression vector. In the case in which the expression vector is a Sendai virus vector, for example, it is suitable to insert the above isolated RNA molecule between a gene-start signal and a gene-end signal, and 1 to 10, 1 to 5, 1 to 3, 2, or 1 isolated RNA molecule(s) as defined above inserted between a gene-start signal and a gene-end signal can be introduced into a single Sendai virus vector.
Introduction of the expression vector according to the present embodiment into cells can be performed by using a well-known method in the art according to the type of cells and expression vectors. For nonviral vectors, for example, introduction can be performed with lipofection, electroporation, or microinjection. For virus vectors, introduction can be performed by infecting cells with an appropriate titer or multiplicity of infection (MOI).
The isolated RNA molecule according to the first embodiment and the expression vector according to the second embodiment can express artificial miRNA/siRNA with a significantly higher efficiency than conventional artificial miRNA/siRNA expression systems, thus being useful.
The present invention will be further described with reference to Examples shown below. These by no means limit the present invention.
1. Expression of Various miRNAs from Sendai Virus Vector
(1-1) Production of miRNA Expression Vector
Various natural miRNA precursors were introduced into Sendai virus (SeV) vectors, and expression levels of miRNAs and gene knockdown effects were evaluated. The SeV vector used was a SeVdp vector (J. Biol. Chem., (2011), Vol. 286, No. 6, pp. 4760-4771). To the downstream of the P/C/V in the SeVdp vector, a selection marker blasticidin resistance gene (blasticidin S deaminase gene; prepared by PCR using a pCX4bsr plasmid (Proc. Natl. Acad. Sci. USA, (2003), Vol. 100, No. 23, pp. 13567-13572) as a template) and an expression marker EGFP gene (prepared by PCR using a pEGFP-1 plasmid (Takara Bio Inc.) as a template) were introduced, and an miR-124 gene or an miR-302-367 cluster was further introduced downstream thereto to prepare a miR-124 expression vector (SeV-124) and a miR-302-367 expression vector (SeV-302-367). The miR-124 gene and miR-302-367 cluster had been prepared by PCR using, as a template, a genomic DNA extracted from C57BL/6J mouse embryonic fibroblasts (MEF). A vector containing no miRNA gene (SeV-Ctrl) was prepared as a negative control.
In addition, a retrovirus vector into which the miR-302-367 cluster had been introduced (Retro-302-367) was prepared in the following procedure. The miR-302-367 cluster cloned from MEF was introduced into the BamHI and NotI sites of a pCX4pur plasmid (Proc. Natl. Acad. Sci. USA, (2003), Vol. 100, No. 23, pp. 13567-13572). HEK293T cells were transfected with the plasmid vector obtained together with pVPack-GP (Agilent Technologies) and pVPack-Ampho (Agilent Technologies) by using FuGENE HD (Promega Corporation). On day 3, the culture supernatant was collected and filtered through a 0.45-μm filter to prepare a miR-302-367 expression retrovirus vector.
(1-2) Quantification of Expression Levels of miRNA
HCT116 cells were infected with the SeV vector at MOI=5, cultured from the next day with supplementing the medium with 10 μg/ml blasticidin S, and cells stably retaining the SeV vector genome were selected. For the retrovirus vector, HCT116 cells were infected with 1×109 copies of the vector in the presence of 4 μg/ml polybrene, cultured with supplementing the medium with 0.2 μg/ml puromycin on day 3, and cells stably expressing the miRNA introduced were selected. From the cells, total RNA was extracted by using the ISOGEN reagent (NIPPON GENE CO., LTD.), and RT-qPCR was carried out for each miRNA by using TaqMan MicroRNA Assays (Applied Biosystems). A TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) was used for the RT reaction, and TaqMan Universal PCR Master Mix II, no UNG (Applied Biosystems), was used for qPCR. The expression level of each miRNA was evaluated by using a ΔΔCt method in which the Cq (quantification cycle) value for each miRNA was normalized with the Cq value for RNU48 (endogenous control gene). The TaqMan MicroRNA Assays used for RT-qPCR are shown in the following.
(1-3) Evaluation of Gene Knockdown Effect of miRNA
To evaluate the gene knockdown effect of each miRNA, a reporter vector was prepared by introducing a target sequence having complete complementarity to a miRNA into the 3′ untranslated region of the RLuc gene in the psiCHECK-2 vector (Promega Corporation) comprising a firefly luciferase (FLuc) gene and a Renilla luciferase (RLuc) gene. FLuc and RLuc are expressed from this reporter vector, and when gene knockdown is caused by a miRNA, only the expression of RLuc decreases. Therefore, the activity of RLuc corrected for the transfection efficiency of the reporter vector can be measured by calculating the relative values of RLuc/FLuc.
Cells prepared in (1-2) were transfected with the above reporter vector by using the Lipofectamine 2000 reagent (Thermo Fisher Scientific). About 22 to 25 hours thereafter, the activities of FLuc and RLuc were measured by using a Dual-Luciferase Reporter Assay System (Promega Corporation), and relative values of the RLuc activity (hereinafter, expressed as “RLuc/FLuc value”) were calculated. As a control, a vector incorporating a scramble sequence which is not targeted by a miRNA, in place of a target sequence for a miRNA, was prepared, and cells were transfected therewith to prepare negative control cells, for which luciferase activities were evaluated in the same manner. Scramble sequences were designed by using siRNA Wizard v3.1 Software (InvivoGen). The gene knockdown effect of each miRNA was evaluated based on the activity of a reporter luciferase RLuc by calculating the relative value of RLuc/FLuc in reporter vector-transfected cells when the RLuc/FLuc value in negative control cells was set to 1.0. The target sequences for miRNAs and the corresponding scramble sequences are shown in the following.
The results are shown in
Expression levels of miR-302a, miR-302b, miR-302c, miR-302d, and miR-367 in human iPS cells (PLOS ONE, (2016), Vol. 11, No. 10, e0164720) were quantified in the same procedure as in (1-2), and compared with those in HCT116 cells into which SeV-302-367 had been introduced. The results are shown in
Furthermore, the expression levels of miR-302a, miR-302b, miR-302c, miR-302d, and miR-367 in the Retro-302-367-introduced HCT116 cells and those in the SeV-302-367-introduced HCT116 cells were compared. The results are shown in
These results suggested the possibility that the expression efficiency of miR-367 from the SeV vector was particularly high.
2. Gene Knockdown Effect of miR-367 Expressed from SeV-367
A SeV expression vector into which only an miR-367 precursor (
The results are shown in
3. Expression of miR-124 from artificial miR-124 precursor based on miR-367 precursor Artificial miR-124 precursors (1) and (2) were designed as artificial miRNA precursors based on the secondary structure of the miR-367 precursor: artificial miR-124 precursor (1) was obtained by replacing the miR-367 sequence with an miR-124 sequence while the secondary structure of the miR-367 precursor was completely retained, and artificial miR-124 precursor (2) was obtained by further modifying artificial miR-124 precursor (1) not to include any mismatch/bulge in the double-stranded miR region while the structure of the miR-367 precursor was retained. The nucleotide sequences of artificial miR-124 precursors (1) and (2) are shown in Table 3, and the secondary structures are shown in
A SeV expression vector into which artificial miR-124 precursor (1) or artificial miR-124 precursor (2) had been incorporated was prepared using the same procedure as in (1-1), the expression vector was introduced into HCT116 cells in the same procedure as in (1-2), and the gene knockdown effects were evaluated in the same procedure as in (1-3).
4. Target Gene Knockdown Effect of Artificial miRNA Expressed by Artificial miRNA Precursor Based on Pre-miR-367 (1)
Artificial miRNA precursors targeting the FLuc gene were produced based on the structures of various natural miRNA precursors in a procedure shown below, and the gene knockdown effects of FLuc-targeting artificial miRNAs expressed thereby were compared.
(4-1) Expression of Artificial miRNA from SeV Vector
FLuc-targeting artificial miRNA precursors mimicking their secondary structures were designed based on the structures of a miRNA precursor described in Non-Patent Document 1 (pre-miR-30), a miRNA precursor described in Non-Patent Document 2 (pre-miR-155), and mouse-derived natural miRNA precursors (pre-miR-367, pre-miR-124, and pre-miR-302a). A sequence completely complementary to a target sequence in an mRNA for FLuc, described by Elbashir et al. (Nature, (2001), Vol. 411, No. 6836, pp. 494-498), was used for the sequence of each artificial miRNA. The nucleotide sequences of the FLuc-targeting artificial miRNA precursors are shown in Table 4, and the secondary structures are shown in
SeV expression vectors, each incorporating any of the artificial miRNA precursors, were prepared using the same procedure as in (1-1), each expression vector was introduced into HCT116 cells using the same procedure as in (1-2), and selection was then carried out with blasticidin S. Furthermore, a pGL3-Control vector (Promega Corporation) comprising a sequence encoding FLuc, and a pRL-TK vector (Promega Corporation) comprising a sequence encoding RLuc were introduced into the cells by using the Lipofectamine 2000 reagent, and activities of FLuc and RLuc were measured about 24 hours thereafter, and relative values of FLuc activity (hereinafter, expressed as “FLuc/RLuc value”) were calculated. The gene knockdown effect of each artificial miRNA was evaluated by calculating the relative value of FLuc/RLuc in the cells introduced with the SeV expression vector incorporating each artificial miRNA when the FLuc/RLuc value was set to 1.0 in cells prepared in the same manner, except that SeV-Ctrl was used in place of a SeV expression vector incorporating the artificial miRNA precursor.
The results are shown in
(4-2) Expression of Artificial miRNA by Plasmid Vector
With use of a pOL1 plasmid in place of a SeV vector, plasmid vectors, each incorporating any of the artificial miRNA precursors shown in
The results are shown in
5. Target Gene Knockdown Effect of Artificial miRNA Expressed by Artificial miRNA Precursor Based on Pre-miR-367 (2)
On the basis of the structure of pre-miR-367, an artificial miRNA precursor mimicking the secondary structure thereof and targeting EGFP was produced, and the gene knockdown effect of the EGFP-targeting artificial miRNA expressed from a SeV vector incorporating the artificial miRNA precursor was evaluated. A sequence completely complementary to a target sequence in an mRNA for EGFP (NCBI: Pr008808666) was used for the sequence of the artificial miRNA. The nucleotide sequence of the EGFP-targeting artificial miRNA precursor is shown in Table 5, and the secondary structure is shown in
A SeV vector incorporating the EGFP-targeting artificial miRNA precursor, a hygromycin resistance gene (hygromycin B phosphotransferase gene, obtained by artificial gene synthesis (GenScript Biotech)), as a selection marker, and a Keima-Red gene (prepared by PCR using a phdKeima-Red-S1 plasmid (Medical & Biological Laboratories Co., Ltd.) as a template), as an expression marker, was prepared using the same procedure as in (1-1). The expression vector was introduced into HCT116 cells using the same procedure as in (1-2), the medium was supplemented with 100 μg/ml hygromycin B from the next day, and cells stably retaining the SeV vector genome were selected. Into the cells obtained, a pEGFP-N1 plasmid (Clontech Laboratories, Inc.) and an E2-Crimson expression plasmid were introduced by using the Lipofectamine 2000 reagent. The next day, fluorescence intensities of EGFP and E2-Crimson were measured by flow cytometry. In addition, measurement of fluorescence intensities was performed in the same manner, except that a SeV expression vector incorporating an FLuc-targeting artificial miRNA precursor was used in place of a SeV expression vector incorporating the EGFP-targeting artificial miRNA precursor (negative control). The gene knockdown effect of EGFP-targeting artificial miRNAs was evaluated by calculating the relative value of the fluorescence intensity of EGFP in E2-Crimson positive cells in the negative control as 1.0. The E2-Crimson expression plasmid had been produced by incorporating an E2-Crimson gene (E2-Crimson gene, prepared by PCR using pE2-Crimson (Clontech Laboratories, Inc.) as a template) downstream of a CMV promoter of pOL1.
The results are shown in
6. Target Gene Knockdown Effect of Artificial miRNA Expressed by Artificial miRNA Precursor Based on Pre-miR-367 (3)
On the basis of the structure of pre-miR-367, an artificial miRNA precursor mimicking the secondary structure thereof and targeting mouse p53 was produced, and the gene knockdown effect of the mouse p53-targeting artificial miRNA expressed from a SeV vector incorporating the artificial miRNA precursor was evaluated. A sequence completely complementary to a target sequence in an mRNA for mouse p53, the sequence described by Dirac and Bernards (J. Biol. Chem., (2003), Vol. 278, No. 14, pp. 11731-11734), was used for the sequence of the artificial miRNA. The nucleotide sequence of the mouse p53-targeting artificial miRNA precursor is shown in Table 6, and the secondary structure is shown in
A SeV-p53-targeting artificial miRNA, being a SeV vector incorporating mouse p53-targeting miRNA precursor (1), a hygromycin resistance gene, and a Keima-Red gene, was prepared using the same procedure as in (1-1). The expression vector was introduced into HCT116 cells using the same procedure as in (1-2), the medium was supplemented with 100 μg/ml hygromycin B from the next day, and cells stably retaining the SeV vector genome were selected. Into the cells obtained, a reporter plasmid obtained by incorporating the mouse p53 target sequence into the 3′ untranslated region of the RLuc gene was introduced using the same procedure as in (1-3), and the gene knockdown effect was evaluated. The mouse p53 target sequence and the corresponding scramble sequence are shown in the following.
The results are shown in
Furthermore, artificial miRNA precursors targeting different sites in the mouse p53 mRNA were produced. The nucleotide sequences of mouse p53-targeting artificial miRNA precursors (2) and (3) are shown in Table 8.
SeV-p53-targeting artificial miRNA (1), SeV-p53-targeting artificial miRNA (2), and SeV-p53-targeting artificial miRNA (3), each being a SeV vector incorporating a mouse p53-targeting artificial miRNA precursor, a blasticidin resistance gene, and EGFP, were prepared using the same procedure as in (1-1). Each expression vector was introduced into HCT116 cells using the same procedure as in (1-2), the medium was supplemented with 10 μg/ml blasticidin from the next day, and cells stably retaining the SeV vector genome were selected. Into the cells obtained, a reporter plasmid obtained by incorporating the full-length open reading frame of mouse p53 into the 3′ untranslated region of the RLuc gene was introduced using the same procedure as in (1-3), and the gene knockdown effects were evaluated.
The results are shown in
7. Production of iPS Cells Using SeV-p53-Targeting Artificial miRNA Precursor
To produce iPS cells, c-MYC is typically introduced in addition to three reprogramming factors: KLF4, OCT4, and SOX2. However, c-MYC is an oncogene, and therefore, a problem arises of risk of promoting tumor formation. With regard to this, it has been reported that use of shRNA targeting p53 enables promotion of iPS cell induction (Nature, (2009), Vol. 460, No. 7259, pp. 1140-1144), and thus, whether iPS cells can be produced by expressing p53-targeting artificial miRNA from a SeV vector, instead of the c-MYC gene, was tested. A SeV vector incorporating a mouse p53-targeting artificial miRNA precursor, a KLF4 gene, an OCT4 gene, and a SOX2 gene was prepared in the same procedure as in (1-1). The KLF4 gene, OCT4 gene, and SOX2 gene had been obtained by artificial gene synthesis (GenScript Biotech). The genomic configurations of the SeV-(KOS) vector and SeV-(mip53/KOS) vector are shown in
SeV-(KOS) and SeV-(mip53/KOS) were introduced into MEF at MOI=5. The next day, cells into which a vector had been introduced (1×104 cells) were placed on MEF treated with mitomycin C and were cultured in mouse ES medium. On day 14, immunostaining was performed by using an antibody against SSEA1 (eBioscience), a pluripotency marker. Cells into which a SeV vector, into which no foreign gene had been introduced (SeV-empty), were used as a negative control.
The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-011541 | Jan 2019 | JP | national |
This application is a 35 U.S.C. § 371 national phase application of PCT Application PCT/JP2020/002519, filed Jan. 24, 2020, which claims priority to Japanese Application No. 2019-011541, filed Jan. 25, 2019, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/002519 | 1/24/2020 | WO | 00 |
